This document discusses pharmacological and analytical interferences that can occur in hormone assays used to diagnose adrenal incidentalomas. It describes several issues that can lead to false hormone concentrations and misinterpretation of results, including circadian rhythms, medical treatments, analytical method limitations, and preanalytical factors like posture and diet. The review focuses on specific interferences that may occur in testing for pheochromocytoma, primary aldosteronism, and other adrenal tumor types. Addressing preanalytical variables, using appropriate reference ranges, and interpreting results in light of comorbidities and medications can help reduce interference effects.

1. Annales d’Endocrinologie 80 (2019) 250–258
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Review
Pharmacological and analytical interference in hormone assays for
diagnosis of adrenal incidentaloma
Interférences pharmacologiques et analytiques des dosages hormonaux pour le
diagnostic des incidentalomes surrénaliens
Antoine-Guy Lopeza,∗
, François Fraissineta
, Herve Lefebvreb,c
, Valéry Brunela
,
Frédéric Zieglera,d
a
Institute for Clinical Biology-General Biochemistry Unit, Rouen University Hospital, 76000 Rouen, France
b
Department of Endocrinology, Rouen University Hospital, 76000 Rouen, France
c
Inserm Unit U1239, Laboratory of Differentiation & Neuronal and Neuroendocrine Communication, IRIB, Normandie University, 76130
Mont-Saint-Aignan, France
d
Inserm U1073, Laboratory of Digestive Tract Environment and Nutrition ADEN, Normandie University, 76000 Rouen, France
a r t i c l e i n f o
Keywords:
Adrenal incidentaloma
Interference
Hormone assays
a b s t r a c t
Adrenal incidentaloma refers to an asymptomatic adrenal mass detected through an imaging procedure
performed for reasons unrelated to adrenal dysfunction or suspected dysfunction. In general, adrenal
incidentalomas are non-functioning adrenal adenomas, but in some cases, may require therapeutic
intervention: eg., adrenocortical carcinoma, pheochromocytoma, primary aldosteronism, cortisol hyper-
secretion, or adrenal insufficiency. Hormone assessment is crucial to characterize adrenal incidentaloma.
Nowadays, various hormone assay methods are available, such as immunoassay and mass spectrome-
try. However, there are several pitfalls that should be considered: e.g., circadian rhythm, gender/age
dependency, preanalytical and analytical issues, and drug interactions. Pharmacological or analytical
interference can lead to false serum concentrations and may result in misinterpretation of results and
thus inappropriate treatment. The purpose of this review was to study the main interferences that may
be observed in the different tumor types of adrenal incidentalomas in order to help physicians in their
clinical decision-making and for the overall benefit of patients.
© 2019 Elsevier Masson SAS. All rights reserved.
Mots clés :
Incidentalomes surrénaliens
Interférences
Dosages hormonaux
r é s u m é
Un incidentalome surrénalien est une masse surrénalienne asymptomatique découverte de façon fortu-
ite par un examen d’imagerie dont l’indication initiale est indépendante d’une pathologie surrénalienne
suspectée. La plupart du temps, il s’agit d’adénomes surrénaliens non fonctionnels, mais dans certains
cas, une intervention thérapeutique est nécessaire (corticosurrénalome, phéochromocytome, hyperal-
dostéronisme primaire, hypersécrétion de cortisol, insuffisance surrénalienne, par exemple). Le bilan
hormonal est essentiel pour caractériser la fonctionalité d’un incidentalome surrénalien. De nos jours,
il existe différentes méthodes de dosages des hormones (techniques immunologiques, spectrométrie
de masse..). Il y a, cependant, certains pièges dont il faut tenir compte : le rythme circadien, des cor-
rélations avec l’âge ou le sexe, certaines questions analytiques ou préanalytiques et les interactions
médicamenteuses. Les interférences pharmacologiques ou analytiques retrouvées dans ces techniques
peuvent être responsables d’erreurs de dosages conduisant à des erreurs d’interprétation, et donc de prise
en charge. L’objectif de cette revue est de présenter aux praticiens les principales interférences pouvant
être observées dans le cadre du bilan biologique des différents types d’incidentalomes surrénaliens, pour
améliorer la décision clinique et la prise en charge des patients.
© 2019 Elsevier Masson SAS. Tous droits réservés.
∗ Corresponding author.
E-mail address: antoine-guy.lopez@chu-rouen.fr (A.-G. Lopez).
https://doi.org/10.1016/j.ando.2018.11.006
0003-4266/© 2019 Elsevier Masson SAS. All rights reserved.

2. A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258 251
1. Introduction
Adrenal incidentalomas are asymptomatic masses detected in
absence of suspected adrenal diseases. The imaging examination
is not executed for symptoms related to adrenal hormone excess,
but rather for the evaluation of symptoms that are not related to
an adrenal disease, eg., back or abdominal pain. Physicians should
perform additional investigations only in lesions ≥ 1 cm [1–4]. The
incidence and prevalence of adrenal incidentalomas can only be
extrapolated from imaging or autopsy studies. Autopsy studies
report a prevalence of around 2% of clinically unapparent adrenal
masses (range 1.0–8.7%) [5,6]. Radiological studies suggest a preva-
lence of around 3% at the age of 50 years which increases with
age (up to 10% in the elderly) [5,7,8]. Adrenal incidentalomas are
extremely rare in childhood. Adrenal incidentalomas are composed
of benign and malignant lesions derived from the adrenal cortex,
the medulla or of extra-adrenal origin (Table 1).
Biological hormone assessment is crucial in characterizing
adrenal incidentaloma. However, there are many pitfalls that
should be considered (e.g., circadian rhythm, gender, age, drug
interactions, preanalytical and analytical issues). Furthermore,
since normal ranges depend on the method used, it is essential
to interpret test results using appropriate reference ranges. Inter-
ferences occur when a substance or process falsely alters an assay
result. Interferences are of pharmacological or analytical origin and
can lead to falsely elevated or falsely low serum analyte concentra-
tions. The consequences of such interferences can be devastating
and may result in the misinterpretation of a patient’s results lead-
ing to a wrong course of treatment. Immunoassay interferences
are most commonly due to antibodies or cross-reaction. They may
be autoantibodies or heterophile antibodies that predominantly
interfere in two-site immunometric (sandwich) assays, between
capture and detection antibodies. The purpose of this review was to
study the main interferences relative to the different tumor types of
adrenal incidentalomas in order to help physicians in their clinical
decision-making and for the overall benefit of patients.
2. Pheochromocytoma
A pheochromocytoma is a tumor arising from adrenal medulla
chromaffin cells that produces catecholamines: adrenaline, nora-
drenaline, and dopamine. In some cases, these tumors can be
biochemically silent. A paraganglioma (PPGL) is a tumor derived
from the ganglia of the sympathetic chain in thorax, abdomen,
and pelvis and from parasympathetic chain in head and skull
base areas. Malignancy in PPGL is characterized by the presence
of metastasis in lymph nodes or other distant sites: bones, lungs,
and liver. The prevalence of PPGL in patients with hypertension
is 0.2% to 0.6% [9], whereas in patients with an incidentaloma,
it increases to 7% [3]. Initial biochemical assessments in case
of clinical suspicion of a pheochromocytoma or PPGL should
include measurements of plasma-free metanephrines or urinary
metanephrines. Metanephrines exist in plasma and urine in
free and mainly in sulfate-conjugated forms [11]. The last data
Table 1
Frequency of the different tumor types of adrenal incidentalomas. (Series including
all patients with an adrenal mass). Adapted from Terzolo M et al. [3].
Tumor entity Median (%) Range (%)
Adenoma 80 33–96
Non-functioning 75 71–84
Autonomously cortisol-secreting 12 1.0–29
Aldosterone-secreting 2.5 1.6–3.3
Pheochromocytoma 7.0 1.5–14
Adrenocortical carcinoma 8.0 1.2–11
Metastasis 5.0 0–18
established the superiority of plasma-free metanephrines because
of better sensitivity and easier sampling conditions. However,
specialized analytical methods as liquid chromatography with
electrochemical detection (LC-ECD) or liquid chromatography with
tandem mass spectrometry (LC-MS/MS), which are not available in
all laboratories, are required. An increase in plasma metanephrines
above 2-fold the upper cut-off suggests that the patient has a
pheochromocytoma [9–11]. The determination of the dopamine
metabolite e.g., 3-methoxytyramine (3MT) (both in urine and
plasma) facilitates the diagnosis of malignant PPGL but can also be
found in patients with neck and skull PPGL. Nevertheless, this test
is not commonly performed in all laboratories [12], and otherwise
may have low specificity [13].
2.1. Sampling conditions
No dietary restrictions are needed for measurements of plasma
metanephrines. Dietary use of amine rich foods might cause
false-positive results for 3MT. Catecholamine-containing products
can be found in bananas, nuts, tomatoes, and beans. Procedures
to avoid dietary influences on metabolites measured are not
commonly taken into account. Therefore, sampling should be
collected after an overnight fast [14,17]. For measurements of
plasma metanephrines, blood should be drawn after 30 minutes
of supine rest [15]. This delay may reduce the risk of miss-
ing a PPGL. The strong influence of sympathetic activation and
upright posture stimulate the release of noradrenaline with sub-
sequent normetanephrine production, leading to an increase of
+30% for normetanephrine, +12% for metanephrine but has no
influence on 3MT levels [16]. Urinary assessment can help when
supine sampling cannot be used. Nevertheless, it is important to
ensure that patients provide a complete 24 h urine collection with
simultaneous measurement of total volume and urinary creatinine
determination [17].
2.2. Physiological or pathological situations
Stress situations associated with acute illness (intensive care,
sepsis, heart failure, hypoglycemia) should be considered in inter-
preting marked elevations of plasma or urine metanephrines.
Such comorbidities accompanied by strong elevations of sym-
pathoneural activity are a source of falsely-elevated plasma
normetanephrine levels [18]. Since an increase in normetanephrine
levels is observed in the elderly, reference values adjusted for age
have been suggested [19,20]. Plasma total metanephrines are also
increased in case of renal insufficiency; taken as a whole, the mea-
surements of plasma-free metanephrines must be preferred [21].
2.3. Medical treatments
There are two kinds of drugs interfering with catecholamine
metabolism and resulting in raised values of biochemical
results due to analytical or pharmacological interferences.
Some drugs directly interfere with measurement methods (e.g.,
acetaminophen, mesalamine and sulfasalazine in high pressure
LC-ECD methods). Moreover, other medications used for hyper-
tension or neurological diseases interfere with the secretion of
catecholamines (e.g., tricyclic antidepressants block the neuronal
uptake of catecholamines). About 40% of interferences are due to
acetaminophen and tricyclic antidepressants, which are the most
common medications in the care of pheochromocytoma [22–24].
To sum up, it is necessary to washout all interfering medications
when possible (Table 2).

3. 252 A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258
Table 2
Major medications that may cause falsely elevated test results for plasma and urinary metanephrines. Adapted from Grouzmann E et al. [25].
Plasma Urine
NMN MN NMN MN
Analytical interferencesa
Acetaminophen, sulfasalazine ++ − ++ −
␣-methyldopa ++ − ++ −
Pharmacological interferences
Increase of catecholamine synthesis
Antiparkinsonian drugs: levodopa + + ++ +
Inhibitor of catecholamine reuptake
Tricyclic antidepressants: amitriptyline ++ – ++ −
Cocaine ++ + ++ ++
Inhibitor of catecholamine catabolism ++ ++ ++ ++
MAO-inhibitors
Increase in catecholamine release
Alpha blocker: phenoxybenzamine ++ – ++ –
Amphetamines: ecstasy + + + +
Sympathomimetics: ephedrine, pseudoephedrine, phenylephrine + + + +
COMT: Catechol-O-methyltransferase; MAO: monoamine oxidase; MN: metanephrine; NMN: normetanephrine; ++: high increase/ + mild increase/-: no increase.
a
These interferences are not observed with the use of chromatography-mass spectrometry methods.
2.4. Analytical issues of measurement methods
The latest studies revealed that measurements of fraction-
ated metanephrines by mass spectrometric methods as LC-MS/MS
have negligible analytical interference compared to high pres-
sure LC-ECD [3]. However, pharmacological interferences are still
possible [25,26].
2.5. Practical approach
In most situations interferences in the biological assessment
of pheochromocytoma are due to inappropriate sampling con-
ditions. This issue is easily dealt with by repeat sampling in
supine position together with stopping medical treatment that
may interfere. When clinical suspicion is low, patients can be
monitored by biochemical follow-up. When clinical suspicion
is moderate or high and plasma normetanephrine is elevated,
clonidine suppression test is useful to exclude the presence of
tumor [27]. However, this test has not been validated in any
prospective study yet. Others have proposed the combination
of measurements of chromogranin A and urinary fractionated
metanephrines as follow-up tests in case of plasma metanephrine
increase [28]. Nevertheless, the use of functional imaging modal-
ities as 123I-meta-iodobenzylguanidine (MIBG) scintigraphy can
help to disprove a diagnosis of pheochromocytoma [29]. It has
been reported that some pheochromocytomas are not associ-
ated with hypertension (normotensive incidentally discovered
pheochromocytomas: NIPs). In approximately 25% of patients,
pheochromocytomas are incidentalomas with normal blood pres-
sure and normal values of metanephrines. In such cases, the CT
scan characteristics of the incidentaloma are important to alert the
clinician [30].
3. Primary aldosteronism
In patients with hypertension or hypokalemia, the latest guide-
lines recommend the use of the aldosterone-renin ratio (ARR)
to exclude primary aldosteronism (PA). Numerous studies have
demonstrated that this ratio is the parameter with the highest
sensitivity (68–94%) and best negative predictive value [31–33].
The ARR has good within-patient reproducibility when performed
under standardized conditions [34]. Like all biochemical investiga-
tions, the ARR is submitted to interferences [35].
3.1. Sampling conditions
The specificity of ARR is better with a sodium-normal diet.
Low-sodium diet elevates renin and plasma aldosterone levels and
lowers ARR [36]. Normal serum potassium concentration is advis-
able because low concentrations inhibit aldosterone secretion and
may induce false negative screening. Aldosterone and renin levels
rise in upright positions in relation with physiological adaptation.
This stimulation is more important in the morning, thus, it is rec-
ommended to collect blood samples in the morning after patients
have been out of bed for at least 2 hours, preferably after they have
been seated for 5–15 minutes [37].
3.2. Physiological or pathological situations
Impaired renal function lowers renin concentration [38]. Dur-
ing pregnancy, ARR and especially plasma aldosterone levels are
increased. Renin secretion decreases with age, probably in relation
with progressive nephron loss. This may lead to false-positive ARR,
and the ARR threshold for PA screening should ideally be revised
after the age of fifty years [39].
3.3. Medical treatments
Many medications affect the ARR. It is necessary to washout
all interfering medications when possible (Fig. 1). For example,
estrogen and progesterone treatment (including oral contracep-
tive agents, hormonal replacement therapy in postmenopausal
women) can induce false-positive ARR elevation if renin is assessed
as “direct renin” concentration (DR). This interference is avoided
with the measurement of plasma renin activity (PRA). Estrogenic
impregnation increases liver angiotensinogen production and thus
angiotensin-II, inhibiting renin secretion by negative feedback with
little change in enzymatic activity. Some authors highlight the
increased precision obtained by the assessment of PRA, which takes
into account individual angiotensinogen concentration, instead of
DR [40–42]. With regard to antihypertensive treatment, it has been
shown that beta-blockers reduce the secretion of renin and plasma
aldosterone. Elsewhere, converting enzyme inhibitor (ACE) and
angiotensin-II receptor antagonist (ARA-II) decrease plasma aldos-
terone and increase plasma renin concentrations; finally, diuretics
induce renin elevation [32] (Table 3).

4. A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258 253
Fig. 1. Medications affecting the aldosterone-renin ratio. ACE: angiotensin-converting enzyme; ARBs: angiotensin II type 1 receptor blockers; ARR: aldosterone-renin ratio;
DHPs: dihydropyridines; FP: false positive; FN: false negative; NSAIDs: nonsteroidal anti-inflammatory drugs; SSRIs: selective serotonin reuptake inhibitors. Adapted from
Douillard C et al. [32] and Funder JW et al. [33].
Table 3
Non-analytical factors that may affect the aldosterone-renin ratio and thus lead to false-positive or false-negative results. Adapted from Funder JW et al. [31].
Factors Effect on aldosterone levels Effect on renin levels Effect on ARR
Medications
␤-Adrenergic blockers ↓ ↓↓ ↑ (FP)
Central ␣-2 agonists (e.g. clonidine, ␣ -methyldopa) ↓ ↓↓ ↑ (FP)
NSAIDs ↓ ↓↓ ↑ (FP)
K+
-wasting diuretics → ↑ ↑↑ ↓ (FN)
K+
-sparing diuretics ↑ ↑↑ ↓ (FN)
ACE inhibitors, ARBs ↓ ↑↑ ↓ (FN)
Ca2+
blockers (DHPs) → ↓ ↑ ↓ (FN)
Renin inhibitors ↓ ↓↑ ↑ (FP)/↓ (FN)a
Serum potassium status
Hypokalemia ↓ → ↑ ↓ (FN)
Potassium loading ↑ → ↓ ↑ (FP)
Dietary sodium
Sodium restricted ↑ ↑↑ ↓ (FN)
Sodium loaded ↓ ↓↓ ↑ (FP)
Other conditions
Advancing age ↓ ↓↓ ↑ (FP)
Renal impairment → ↓ ↑ (FP)
PHA-2 → ↓ ↑ (FP)
Pregnancy ↑ ↑↑ ↓ (FN)
Renovascular HT ↑ ↑↑ ↓ (FN)
Malignant HT ↑ ↑↑ ↓ (FN)
ACE: angiotensin-converting enzyme; ARBs: angiotensin II type 1 receptor blockers; ARR: aldosterone-renin ratio; DHPs: dihydropyridines; FP: false positive; FN: false
negative; HT: hypertension; NSAIDs: nonsteroidal anti-inflammatory drugs; SSRIs: selective serotonin reuptake inhibitors.
a
Renin inhibitors lower plasma renin activity (PRA) but raise direct renin concentration (DRC). This is expected to result in false-positive ARR levels for renin measured as
PRA and false-negatives for renin measured as DRC.
3.4. Analytical issues of measurement methods
Plasma aldosterone may be measured by radioimmunology
(RIA) or chemiluminescence, the most common technique, or by
LC-MS/MS, the latter method being already developed in several
laboratories for its better sensitivity and specificity [43]. RIA meth-
ods undergo interferences from polar metabolites of aldosterone
(tetrahydroaldosterone and aldosterone-18-glucuronide) [44]. In
addition, plasma aldosterone measurements by immunoassay in
patients with renal impairment can be overestimated due to an
increase in aldosterone metabolites, which cross-react with reac-
tive antibodies [45]. Renin may be assayed in two ways. PRA is
assessed by RIA, but it is a manual and time-consuming method.
Plasma renin concentration, also known as DR immunoassay is
now the most common renin assay because of fast standardized
analysis using an automated analyzer, with lower production costs
[46]. Besides, the cut-off of ARR depends on the measurement
method and the unit measure of renin and aldosterone. If standard-
ized assay conditions are respected, the ARR threshold to screen
PA is about 64 according to the latest guidelines (aldosterone in
pmol/L, direct renin in mIU/L) (conversion factors: aldosterone:
pmol/L = pg/mL × 2.77 and direct renin: mIU/L = pg/mL × 1.67) [32].
However each laboratory should check its own cut-off [47].
3.5. Practical approach
In most situations, interferences in renin and aldosterone assays
are due to inappropriate sampling and can be dealt with by repeat
sampling in standardized conditions. To avoid overestimating ARR
generally due to a very low renin concentration, a value of 5
mIU/L should be attributed to any direct renin result < 5 mlU/L
[32]. With regard to blood sampling, standard conditions should be
followed [32]:

5. 254 A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258
• in the morning (out of bed for at least 2 hours);
• seated for 5–15 minutes;
• under normal sodium diet/encourage patient to liberalize sodium
intake;
• urinary sodium,100–200 mmol/24 h;
• potassium-replete/with normal serum potassium values/correct
hypokaliemia;
• washout of all interfering medications (anti hypertensive/
estrogens-progestins).
Finally, when screening by ARR reveals conflicting results
between plasma aldosterone and renin concentrations, dynamic
tests as saline infusion may be undertaken in these ambiguous cases
to confirm autonomous aldosterone secretion [32].
4. Cushing’s syndrome
Cushing’s syndrome (CD) is characterized by excessive cortisol
secretion from the adrenocortical gland. The latest guidelines rec-
ommend that all patients with adrenal incidentalomas undergo in
first line a 1 mg overnight dexamethasone suppression test (DST) as
a screening test to exclude cortisol hypersecretion [48,49]. A value
≤ 50 nmol/L (≤1.8 ␮g/dL) is considered as normal, allowing exclu-
sion of cortisol hypersecretion [48]. To confirm cortisol secretory
autonomy, some additional biochemical tests should be performed,
such as 24 h urinary free cortisol (UFC), midnight salivary cortisol
(MSC) assay repeated at least 2 times. However, the recent pub-
lished literature is controversial and no clear statement has been
made on these tests in patients with adrenal incidentaloma [48].
4.1. Pre-analytical conditions, physiopathological situations and
medical treatments
Plasma cortisol is 80% bound to cortisol-binding globulin (CBG)
and 10–15% to albumin. Some disorders can reduce (inflammation,
rare genetic disorders) or increase CBG levels (estrogen, pregnancy,
mitotane). Since most cortisol is bound to CBG, total serum corti-
sol levels are significantly affected by variation in CBG levels [50].
For example, patients with nephrotic syndrome, liver disease or
malnutrition may have lower CBG levels, with a decrease in corti-
sol levels. Conversely, the use of oral estrogen contraceptives may
result in increased CBG levels, leading to high serum cortisol con-
centrations. Whenever possible, estrogen-containing drugs should
be withdrawn for 6 week before cortisol assessment [51]. Normal
ranges vary substantially, depending on the method used, so it
is essential to interpret test results in the context of the appro-
priate normal range. Hence, immunoassays may be affected by
cross-reactivity with cortisol metabolites or synthetic glucocor-
ticoids. In order to avoid cross-reactions and whenever possible,
patients should be instructed to avoid any glucocorticoid intake
for at least one week before each kind of cortisol sample collection
(blood, urine collection and saliva test). Finally, cortisol hypersecre-
tion may be unrelated to any neoplasia, defined as non-neoplastic
hypercortisolism. These “functional” hypercortisolism are induced
by chronic hypersecretion of ACTH. These physiopathological
situations include: alcoholic impregnation, neuropsychiatric dis-
order, chronic kidney disease, multiple sclerosis, and situations of
insulin resistance (obesity, polycystic ovary syndrome and type 2
diabetes) [52].
4.1.1. Dexamethasone suppression test
The overnight test is easy to implement. Various doses of dex-
amethasone have been proposed, however, 1 mg dexamethasone
is usually given between 11 pm and 12 pm, and cortisol is mea-
sured between 8 am and 9 am the following morning. Variations in
the absorption and metabolism of dexamethasone may influence
the result of the overnight 1 mg dexamethasone suppression test
(DST). Dexamethasone clearance may be reduced in patients with
liver and/or renal failure. The measurement of plasma dexametha-
sone concentration may help to ensure adequate dexamethasone
level, with low specificity [Cut-off > 5.6 nmol/L (0.22 g/dL)] [53].
Conversely, some drugs may induce hepatic enzymatic clearance
of dexamethasone (mediated by CYP 3A4) leading to a decrease
in dexamethasone concentrations, such as phenytoin, phenobarbi-
tone, carbamazepine, rifampicin, and alcohol (Table 4).
4.1.2. Urinary free cortisol assay
Despite the high sensitivity of 1 mg DST in identifying cortisol
status, the low specificity of this test leads to consideration of UFC
levels. UFC provides an integrated assessment of cortisol secretion
over a 24 h period. It is important to ensure that patients provide a
complete 24 h urine collection with appropriate total volume and
to measure urinary creatinine levels. Since UFC is altered by renal
filtration, UFC assessment is not indicated when creatinine clear-
ance falls below 60 mL/min. Hence, UFC levels fall linearly together
with the severity of renal failure [54].
4.1.3. Midnight salivary cortisol assay
Since UFC may be normal in some patients with mild Cush-
ing’s syndrome, salivary cortisol may be more useful in those
cases. Patients are asked to collect a saliva sample on two sepa-
rate evenings between 11 p.m. and 12 p.m. Saliva is collected either
by passive drooling into a plastic tube or by placing a cotton pled-
get (“salivette”) in the mouth and chewing for 1–2 min. In healthy
individuals with stable conventional sleep-wake cycles, the level of
serum cortisol follows a circadian rhythm. Cortisol rises at 3–4 a.m.,
reaches a peak at 7–9 a.m., and then falls to low levels for the rest
of the day. An increase in blood cortisol is reflected by a change in
salivary cortisol concentrations within a few minutes [55]. The loss
of this circadian rhythm can lead to a diagnosis of Cushing’s syn-
drome. It is important to note that the circadian rhythm is blunted
in many patients with depressive illness and in shift workers [56].
Cigarettes smokers have been shown to exhibit higher late-night
salivary cortisol levels than non-smokers. Although the duration of
this effect is not known, it is advisable to avoid cigarette smoking
on the day of saliva collection [57]. Moreover, abnormal elevated
midnight salivary cortisol assay levels have been reported in elderly
subjects and in those with diabetes mellitus [58]. Various methods
have been used to measure cortisol in the saliva, resulting in dif-
ferent reference ranges and yielding differences in sensitivity and
specificity.Accordingto the low concentrationsmeasured,the best-
validated assay is LC-MS/MS rather than RIA or chemiluminescence
assays so far [59].
4.2. Analytical issues of measurement methods
The immunoassays most commonly used in some laboratories
are affected by several limitations, especially those that require
proper extraction and prepurification. High-pressure liquid chro-
matography (HPLC) or liquid chromatography tandem mass
spectrometry assays are highly specific (especially LC-MS/MS) and
provide reliable measurements of cortisol in plasma, urine and sali-
vary samples. Immunoassays without extraction can be affected by
cross-reaction with cortisol metabolites and synthetic glucocorti-
coids. In contrast, molecular structural based assays as HPLC and
LC-MS/MS avoid this problem and are being used with increasing
frequency [59]. However, there are also drugs (carbamazepine and
fenofibrate) that may interfere with some chromatographic meth-
ods of free urinary cortisol assessment causing falsely elevated
values [60]. In patients treated with metyrapone, the block effect
on 11␤-hydroxylase induces an increase in 11 ␤-desoxycortisol,
which may result in positive interference with cortisol

6. A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258 255
Table 4
Selected drugs that may interfere with the interpretation of biological results in the diagnosis of Cushing’s syndrome. Adapted from Nieman LK et al. [49].
Drugs that accelerate
dexamethasone metabolism by
induction of CYP3A4
Drugs that impair
dexamethasone metabolism by
inhibition of CYP3A4
Drugs that increase cortisol
binding globulin and may
falsely elevate cortisol results
Drugs that increase urinary
free cortisol results
Pharmacological interferences Pharmacological interferences Pharmacological interferences Pharmacological interferences
Phenobarbitone Aprepitant/fosaprepitant Estrogens Drugs that inhibit 11␤-
Phenytoin Itraconazole Mitotane HSD2 (licorice, carbenoxolone)
Carbamazepine Ritonavir
Primidone Fluoxetine Analytical interferences
Rifampin Diltiazem Carbamazepine
Rifapentine Cimetidine Fenofibrate (LC-MS/MS)
Ethosuximide Glucocorticoids (Immunoassays)
Pioglitazone
LC-MS/MS: liquid chromatography with tandem mass spectrometry.
measurement when using some immunoassay, due to the very
similar molecular structure of both steroids. In this case, analysis
using mass spectrometry would be preferable [61]. Relative to the
same mechanism, the upper limits of normal ranges are lower with
HPLC or LC-MS/MS than with immunoassays, which may measure
additional compounds with very similar molecular structures. To
sum up, immunoassays should be preferred for serum cortisol
assessment in emergency cases, and LC-MS/MS should be used for
its high specificity when interferences are suggested or in specific
situations as pregnancy.
4.3. Practical approach
Each cortisol test has its drawbacks and the choice of test should
be individualized for each patient. UFC determination should not
be used in patients with renal impairment. With regard to the
1 mg DST test, dexamethasone concentration may be measured
in some difficult situations; if the concentration is too low at 8–9
am, the test should be repeated using a higher dose of dexametha-
sone to achieve an accurate morning level [49]. The measurement
of serum CBG concentration could be of interest in patients with
estrogenotherapy, but it’s not usually used in routine. In case of an
increase above the normal values, estrogen should be discontinued
for 4–6 weeks [49]. Finally, if salivary cortisol is used in specific pop-
ulations such as aged and diabetic patients (> 60 years old), night
shift workers or cigarettes smokers, only normal results should be
validated [49].
4.4. Adrenocortical carcinoma
Adrenocortical carcinoma (ACC) is less frequent but accounts
for 2-11% of incidentally discovered adrenal tumors. The European
Network for the Study of Adrenal Tumors (ENS@T) suggested a
pre-operative hormonal biological assessment in case of suspected
ACC. It includes assessment of dehydroepiandrosterone sulfate,
17-hydroxyprogesterone, androstenedione, testosterone, and 17-
beta-estradiol (only in men and postmenopausal women) [62]. All
steroid hormones are derived from cholesterol and share a basic
cyclopentano-perhydrophenanthrene cycle structure. The small
structural differences, unique to each steroid hormone (modifica-
tions to the basic 4-cycle steroid structure) explain why it is difficult
to discriminate each steroid using a direct immunoassay method.
This analytical interference is known as cross reaction. The best way
to overcome these issues is to use LC-MS/MS. A promising approach
for differentiating adenomas from ACCs uses mass spectrometry-
based steroid profiling of 24 h urine samples. Some recent studies
have suggested that urine steroid metabolomic analysis could be
a new tool to discriminate benign from malignant adrenocorti-
cal tumors, but this method has not yet been designed for routine
use [63].
5. Adrenal insufficiency
In rare cases, adrenal incidentaloma can be bilateral. Bilat-
eral adrenal enlargement due to metastatic diseases or adrenal
hemorrhages can lead to adrenal insufficiency. Adrenal insuffi-
ciency is defined by the inability of the adrenal cortex to produce
sufficient amounts of cortisol. It is a severe and potentially life-
threatening disease due to the central role of these hormones in
energy, salt, and fluid homeostasis. Primary adrenal insufficiency
(PAI), also known as Addison’s disease, should be distinguished
from secondary adrenocortical insufficiency due to insufficient
production of adrenocorticotropic hormone (ACTH) and without
impact on the renin angiotensin-aldosterone system. Patients with
autoimmune disease, systemic disorders or situations of increased
cortisol metabolism induced by drugs have an increased risk of
PAI. The diagnosis of adrenal insufficiency is based on low morn-
ing cortisol concentrations (<138 nmol/L (5 ␮g/dL)) and confirmed
by low stimulation of cortisol secretion by corticotropin [64,65].
The corticotropin stimulation test (or short Synacthen standard
dose: 250 ␮g) test is currently considered as the gold standard for
the diagnosis of primary adrenal insufficiency. Usually, a peak of
cortisol concentration after acute stimulation with corticotropin
exceeding 500 nmol/L (18 ␮g/dL) is accepted as evidence of suffi-
cient adrenocortical activity [65]. However, those cut-offs (morning
cortisol level and after stimulation with corticotropin) are still
debated and it is difficult to set up the best range [66,67]. In prac-
tice, since adrenal insufficiency can be a life-threatening disease, a
higher threshold has been proposed to avoid eliminating a diagno-
sis of partial adrenal insufficiency (false negative) [64]. Otherwise,
insulin tolerance test should be preferred for secondary adrenal
insufficiency [65]. Dehydroepiandrosterone sulfate (DHEA-S) lev-
els that are below the lower limit of normal range (adjusted for
gender and age) may be a useful sign of PAI [65,66].
5.1. Pre-analytical conditions
Since cortisol base-line value is modulated by stress, physical
activity and food, fasting and a period of rest of at least 15 minutes
should be recommended before blood cortisol sampling. Since the
peak level of cortisol is seen in the early morning, sampling should
be performed at this time of day (between 6 am and 10 am) to
detect a reduction of cortisol synthesis [65].
5.2. Physiological or pathological situations
There are several conditions requiring specific considerations
since cortisol level is expected to be altered by nonadrenal patholo-
gies, as critical illness and pregnancy. Reduced CBG levels in illness
and elevated levels in pregnancy may alter the interpretation of cor-
tisol levels [50]. The use of estrogen-containing oral contraceptives
results in higher CBG with a corresponding rise in cortisol. Patients

7. 256 A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258
Table 5
Main origins of variations in corticosteroid binding globulin.
Variations of serum
CBG concentration
Drugs Physio-pathological conditions
Estrogens
Mitotane
Antiepileptic
drugs
Pregnancy/estrogenotherapy
Hepatitis
Corticosteroid
Interleukin-6
Inflammation
Cushing’s syndrome
PCOS
Cirrhosis
Hypothyroidism
Nephrotic syndrome
CBG deficiency disease
CBG: corticosteroid binding globulin; PCOS: Polycystic ovary syndrome.
with nephrotic syndrome and liver disease as well as those who
are in immediate postoperative period or who require intensive
care may have lower CBG and albumin and hence, lower cortisol
measurements (Table 5).
5.3. Medical treatments
Immunoassays are more subject to interferences because of
a lack of full specificity of the antibodies used. Several chem-
ical structures that are similar to cortisol have been described
to induce false-positive results (cross-reactivity). Those treat-
ments include prednisolone, cortisone, and 6-methylprednisolone
[68]. In case of pharmacological interferences, new drug classes
actually used in the treatment of melanoma, renal cell carci-
noma and non-small-cell lung cancer may also interfere in blood
cortisol assessment. There are two kinds of cancer immunother-
apy: anticytotoxic anti-T-cell antigen-4 antibodies (anti-CTLA4)
or anti-programmed cell death-protein1 antibodies (anti-PD1).
These antibodies are directed against inhibitory and co-stimulatory
molecules to activate the immune system, in order to supress
tumor cells [69,70]. Immune checkpoint blockade can induce
endocrinopathies, including hypophysitis, thyroid dysfunction and
diabetes mellitus. The incidence of secondary adrenal insufficiency
by hypophysitis related to CTLA4 antibody therapy is around 6%
[71]. Some cases of ipilimumab-induced primary adrenalitis have
been reported but seem to be exceptional [72,73]. Up to now,
there are no data on analytical interference in cases of cancer
immunotherapy. Physicians should report to the laboratory any
discordance between clinical and laboratory data.
5.4. Analytical issues of measurement methods
The majority of laboratories in Europe use routine immunoas-
says with automated methods. Nevertheless, for some specific
situations, methods based on chromatographic separation and
mass spectrometry are suitable. During pregnancy LC-MS thresh-
old levels have been proposed for morning cortisol determination,
e.g., 300 nmol/L (108 ng/mL) in the first trimester, 450 nmol/L
(162 ng/mL) in the second and 600 nmol/L (216 ng/mL) in the third
trimester [74].
5.5. Practical approach
Diagnosis of primary adrenal insufficiency can be difficult
mainly in situations modulated by stress. First of all, it is important
to check that blood cortisol sampling is done in calm conditions
and confirm or exclude diagnosis by corticotropin stimulation test
(short Synacthen standard dose: 250 ␮g). If a cross reaction by cor-
ticosteroid treatment is suspected, clinicians should investigate all
medications given to the patient in the last months. Finally, LC-MS
method may be used in cases with discordant data or in pregnant
patients [75]. The measurement of serum free cortisol and CBG can
also be used but these immunoassays are not routinely available.
Morning salivary cortisol levels may be measured but there are
insufficient data to propose accurate recommendations.
6. Conclusion
Interferences in immunoassays may lead to the misinterpre-
tation of patients’ results possibly leading to a wrong course of
treatment. Laboratories should improve the processes to detect,
test and report suspected analytical interferences. At the same
time, it is important that physicians communicate to the laboratory
any clinical suspicion of discordance relative to patients’ preana-
lytical status and treatment in order to avoid any misinterpretation
Fig. 2. Items that should be considered when inadequate results of hormonal biological assays are suspected. *
Liquid chromatography followed by tandem mass spectrometry.

8. A.-G. Lopez et al. / Annales d’Endocrinologie 80 (2019) 250–258 257
of biological results. The detection of interference may require the
use of an alternate analysis method or additional measurements,
including dilution of the sample in a non-immune serum. Likewise,
it is imperative that laboratories inform physicians of the follow-
up procedure and report on the presence of any interference
(Fig. 2). Finally, a permanent laboratory-physician collaboration
is essential to ensure optimal clinical decision making based on
pharmacological and analytical assays without interferences.
Disclosure of interest
The authors declare that they have no competing interest.
Acknowledgments
The authors are grateful to Nikki Sabourin-Gibbs, Rouen Univer-
sity Hospital, for her help in editing the manuscript.
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